Callitriche Cophocarpa (Water Starwort) Proteome Under Chromate Stress: Evidence for Induction of a Quinone Reductase

Environmental Science and Pollution Research (2018) 25:8928–8942 https://doi.org/10.1007/s11356-017-1067-y

RESEARCH ARTICLE

Callitriche cophocarpa (water starwort) proteome under chromate stress: evidence for induction of a quinone reductase

Paweł Kaszycki1 & Aleksandra Dubicka-Lisowska1 & Joanna Augustynowicz2 & Barbara Piwowarczyk2 & Wojciech Wesołowski3

Received: 16 May 2017 /Accepted: 18 December 2017 /Published online: 13 January 2018 # The Author(s) 2018. This article is an open access publication

Abstract Chromate-induced physiological stress in a water-submerged macrophyte Callitriche cophocarpa Sendtn. (water starwort) was tested at the proteomic level. The oxidative stress status of the plant treated with 1 mM Cr(VI) for 3 days revealed stimulation of peroxidases whereas catalase and superoxide dismutase activities were similar to the control levels. Employing two-dimensional electrophoresis, comparative proteomics enabled to detect five differentiating proteins subjected to identification with mass spectrometry followed by an NCBI database search. Cr(VI) incubation led to induction of light harvesting chlorophyll a/b binding protein with a concomitant decrease of accumulation of ribulose bisphosphate carboxylase (RuBisCO). The main finding was, however, the identification of an NAD(P)H-dependent dehydrogenase FQR1, detectable only in Cr(VI)-treated plants. The FQR1 flavoenzyme is known to be responsive to oxidative stress and to act as a detoxification protein by protecting the cells against oxidative damage. It exhibits the in vitro quinone reductase activity and is capable of catalyzing two-electron transfer from NAD(P)H to several substrates, presumably including Cr(VI). The enhanced accumulation of FQR1 was chromate-specific since other stressful conditions, such as salt, temperature, and oxidative stresses, all failed to induce the protein. Zymographic analysis of chromate-treated Callitriche shoots showed a novel enzymatic protein band whose activity was attributed to the newly identified enzyme. We suggest that Cr(VI) phytoremediation with C. cophocarpa can be promoted by chromate reductase activity produced by the induced quinone oxidoreductase which might take part in Cr(VI) → Cr(III) bioreduction process and thus enable the plant to cope with the chromate-generated oxidative stress.

Keywords Macrophyte . Aquatic plants . Hexavalent chromium . Biological reduction . Phytoremediation . Oxidative stress . Quinone dehydrogenase

Responsible editor: Elena Maestri Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-017-1067-y) contains supplementary material, which is available to authorized users. Anthropogenic pollution with Cr compounds has become a worldwide problem due to extensive use of this metal in a * Paweł Kaszycki number of industrial applications and vehicular transport [email protected] (Shadreck and Mugadza 2013; Singh et al. 2013; Zayed and Terry 2003). Chromium may appear at various oxidation 1 Unit of Biochemistry, Institute of Plant Biology and Biotechnology, states forming chemical agents of different toxicities, solubil- Faculty of Biotechnology and Horticulture, University of Agriculture ities, and stabilities (Kotaś and Stasicka 2000; Zayed and in Kraków, al. 29 Listopada 54, 31-425 Kraków, Poland Terry 2003), and among these, chromate (Cr(VI)) is the most 2 Unit of Botany and Plant Physiology, Institute of Plant Biology and hazardous to living organisms (Saha et al. 2011;Zhitkovich Biotechnology, Faculty of Biotechnology and Horticulture, 2− 2011). Chromate ion (CrO4 ) structurally resembles the sul- University of Agriculture in Kraków, al. 29 Listopada 54, fate anion (SO 2−) and therefore it becomes actively incorpo- 31-425 Kraków, Poland 4 rated by cells mainly through non-specific sulfate transporter 3 Unit of Genetics, Plant Breeding and Seed Science, Institute of Plant systems and in the lesser extent through HPO 2− carriers Biology and Biotechnology, Faculty of Biotechnology and 4 Horticulture, University of Agriculture in Kraków, al. 29 Listopada (Cervantes et al. 2001; Prado et al. 2016; Singh et al. 2013). 54, 31-425 Kraków, Poland Then, inside cells, Cr(VI) as a strong oxidant interacts with Environ Sci Pollut Res (2018) 25:8928–8942 8929 cell constituents and undergoes rapid reduction due to both knowledge on Cr bioconversion mechanisms and response enzymatic and non-enzymatic reactions (Cervantes et al. strategies. 2001; Chandra and Kulshreshtha 2004; Shanker et al. 2005, The mechanisms of chromate uptake, accumulation, and 2009). Chromate bioreduction is in fact a gradual and complex metabolism have been studied for terrestrial plants more thor- route involving formation of Cr(V) and Cr(IV) intermediates oughly than for macrophytes (Shanker et al. 2005, 2009), responsible for generation of reactive oxygen species (ROS). although the latter group seems to be a better choice for In plants, many of these byproducts lead to adverse stress phytoremediation purposes (Tel-Or and Forni 2011). This is effects by causing severe physiological disorders and oxida- because aquatic plants, especially the water-floating and sub- tive damage (Oliveira 2012; Panda and Choudhury 2005; merged species, usually exhibit higher accumulation potential Singh et al. 2013). and have evolved distinct physiological and biochemical path- For the above reasons, Cr decontamination actions need to ways due to their larger contact area with the surrounding be undertaken. Among various methods implemented to date water environment (Chandra and Kulshreshtha 2004). In con- environmentally, bioremediation and especially sequence, the assimilative organs of macrophytes are affected phytoremediation appear as the most promising approaches directly by the solution and this interaction becomes particu- (Parvaiz 2016). In biotechnological applications, plants capa- larly important in the case of heavy metal presence. ble of enhanced chromate uptake and accumulation as well as Several aquatic plants have been proposed as efficient of its reduction to the less toxic forms are of particular interest phytoremediators of Cr contamination (Prado et al. 2016; since they bring possibilities for elaborating cheap, non-inva- Singh et al. 2013, and the references therein). Among these sive, and efficient industrial-scale methods for soil reclama- is the recently studied genus Callitriche that appears as a suit- tion and water cleanup (Prado et al. 2016;Yangetal.2005; able candidate for environmental biotechnological use Zayed and Terry 2003). However, biological remediation of (Augustynowicz et al. 2014b; Favas et al. 2012, 2014). In environmental contamination with chromium compounds in- particular, Callitriche cophocarpa Sendtn. (water starwort), volves complex and divergent processes (Chandra and a widespread species growing both in stagnant and running Kulshreshtha 2004; Panda and Choudhury 2005;Shanker waters, was shown to reveal unusual response to chromate et al. 2005, 2009). In the case of higher plants, chromium proving to be a potent Cr phytoremediator and accumulator stress negatively affects cellular metabolism at different func- (Augustynowicz et al. 2010, 2013a, 2013b, 2016). It exhibited tional levels triggering variant reactions that enable some spe- enhanced capability of both Cr(VI) and Cr(III) cies to adapt to this heavy metal, resist its action, or detoxify phytoextraction from contaminated waters and extremely high hazardous intermediates (Prado et al. 2016; Shanker et al. capacity to bind Cr (3900 mg kg−1 for 5-day treatment at 2009; Zayed and Terry 2003). 1 mM Cr(VI)). Furthermore, it revealed extraordinary poten- Chromate detoxication and/or protective mechanisms in tial in terms of chromate accumulation rate (up to 1.8% of dry plants are complex, may involve multiple factors, and can mass which makes it a Cr hyperaccumulator), intratissular become manifested at different organizational levels related bioreduction kinetics (a postulated detoxication pathway), to plant metabolism, physiology, development, or structure. and bioconcentration factor (BCF determined as 74 for a 5- For the above reasons, they are not yet fully explained and day treatment of the shoots with 1 mM Cr(VI), the value close understood (see Prado et al. 2016 for a recent critical review). to that of commercially used sorbents). Upon incubation with Moreover, these processes differ depending on the strategy 50-μMchromate,C. cophocarpa accumulated > 0.1% of Cr used by particular species to cope with the metal toxicity. with no noticeable changes in the plant physiological status. Based on these divergent strategies, plants can be categorized All the above characteristics imply that the macrophyte has as Cr excluders, indicators, or accumulators (Dalvi and evolved efficient adaptive mechanisms against chromate Bhalerao 2013). For the case of Cr-accumulating and Cr- stress and can respond to Cr(VI) treatment atypically. tolerant plants, it is well evidenced that the chromate stress So far, little has been done with regard to the studies of reaction involves induction of oxidative stress response sys- chromate-induced response of macrophytes at the level of tems. This is achieved by enhancing the activities of antioxi- detectable proteomes. Such research is of high interest, espe- dant enzymes (superoxide dismutase (SOD), catalase (CAT), cially for the case of submerged plants that can interact with peroxidase, glutathione reductase, ascorbate peroxidase, Cr compounds directly via their shoots. Therefore, besides dehydroascorbate reductase, monodehydroascorbate reduc- particular applicability of C. cophocarpa for tase, as well as several others, Ovečka and Takáč 2014; phytoremediation of aquatic systems, this species can serve Shanker et al. 2005; Singh et al. 2013)aswellasbystimulat- as a good model to study chromium effect on cells and tissues. ing bioreduction of Cr(VI) to the final Cr(III) form considered The main aim of the work is to reveal whether chromate treat- more stable and less toxic (see discussion below). The de- ment alters a protein profile of water starwort as detected with scribed processes may, in turn, result in altered proteomic two-dimensional electrophoresis. Then, based on identifica- profiles whose detailed analysis is expected to broaden our tion of the differentiating proteins with mass-spectrometry 8930 Environ Sci Pollut Res (2018) 25:8928–8942 and bioinformatic tools, an attempt was made to establish Other stress conditions whether the observed changes are specific to Cr(VI) and if they can account for the abovementioned unusual Temperature stress was introduced by 24-h incubation in the C. cophocarpa phytoremediation properties. MS medium at 33 °C, then 48 h at control conditions. Salt stress was exerted by incubation in 150-mM NaCl for 24 h, then in MS medium for 48 h. Oxidative stress was applied Materials and methods either by 5-min incubation with 0.1-mM paraquat (methylviologen) or 5-min treatment with 1-mM hydrogen Plant material peroxide and then cultivation in the MS medium for 72 h. Note that all the applied stress-inducing factors had earlier For the study, C. cophocarpa Sendtn. was grown under been thoroughly tested at variant concentrations and treatment in vitro conditions. The source plant material had earlier been times. This enabled to obtain conditions providing consider- collected from its natural habitat: the Dłubnia river located in able stress but not lethal to the plant (data not shown). All the Southern Poland (N 50° 15′ 58″/E 19° 56′ 24.9″)during incubations were performed in a phytotron chamber under the spring of 2012 (May). Healthy, undamaged shoots were rinsed conditions described above. It was assumed that a 72-h incu- for 10 min with a running, tap water. Next, the material was bation time was necessary for Callitriche to evolve the stress- surface-sterilized in 70% ethanol for 25 s, followed by im- like phenotype upon the tested factors, which was based on mersing in 0.5% aqueous solution of sodium hypochlorite another study of Lemna minor L. time-dependent response to for 2 min, and finally, rinsing five times with the sterile dis- stress agents (Forni et al. 2012). After incubations, the plants tilled water. Sterilized shoot fragments with apical buds (5– were washed thrice in distilled water and kept frozen at − 10-mm length) were placed in a liquid MS medium 20 °C. (Murashige and Skoog 1962) composed of macro- and micro- elements supplemented with vitamins and 1% sucrose. Every Preparation of protein samples for gel electrophoresis 6–8 weeks, the young shoots were transferred to a fresh medium. Plants were grown in a phytotron chamber (model FD Because of the relatively low protein concentration found for 500, Biosell, Poland) under the following conditions: 16 h of Callitriche tissues and very high amount of interfering com- photon flux density of 40 μmol m−2 s−1 and 8 h of darkness, at pounds, especially phenolics and their glycosides 22-°C day/18-°C night. Light spectrum and intensity were (Augustynowicz et al. 2014a), the total plant material content similar to that applied in previous studies (Augustynowicz for electrophoretic analyses was extracted using the method of et al. 2016) and were found sufficient for normal plant growth phenol-SDS buffer extraction without sonication, elaborated resembling the conditions typical of Callitriche natural by Chatterjee et al. (2012) with some necessary modifications. environment. One gram of shoots was ground with liquid nitrogen in a mortar and extracted with 3 ml of homogenization buffer con- Plant incubation with chromate taining 30% sucrose, 2% SDS, 0.1-M Tris-HCl, 5% β- mercaptoethanol, and 1-mM PMSF (phenyl methyl sulfonyl Two grams of C. cophocarpa shoots was typically incubated fluoride), pH 8.0. Then, 3 ml of Tris-buffered phenol was for 72 h in 100 ml of MS basal salt medium supplemented added and the mixture vortexed for 15 min at 4 °C, then 2− with 1-mM chromate (CrO4 ) applied by dissolving potassi- centrifuged at 8000 ×g at 4 °C for 15 min. The phenolic frac- um chromate K2CrO4 10-mM stock solution directly into the tion was collected, 3 ml of a homogenization buffer added plant medium (the chemical formula of potassium chromate, again, and the procedure of shaking and centrifugation was given as canonical SMILES is as follows: [O repeated. The phenolic phase was collected, centrifuged, and −][Cr](=O)(=O)[O−]·[K+]·[K+]; for detailed information see precipitated with 0.1-mM ammonium acetate in cold metha- compound summary for CID 24597 in the PubChem Open nol at − 20 °C overnight. Then, the mixture was centrifuged at Chemistry Database, https://pubchem.ncbi.nlm.nih.gov/ 10,000 ×g for 30 min at 4 °C and the pellet washed three times compound/24597#section=Top; accession date: November with 0.1-mM ammonium acetate in cold methanol and once 2017). Time, temperature, and light regimes of cultivation with 80% cold acetone. The resultant precipitate was dried at were as described above. Such conditions were established room temperature and kept frozen at − 80 °C. to cause cellular stress but were not lethal and upon removing Cr(VI) from the medium the plants fully SDS-PAGE electrophoresis recovered (see BResults^). In control experiments, the same amount of plant material was incubated in the MS medium. Protein pellets were solubilized and denatured with a loading Morphological observations of C. cophocarpa shoots were buffer (0.5-M Tris-HCl, pH 6.8, 10% SDS, 10% β- carried out with a binocular microscope Nikon SMZ 1500. mercaptoethanol, 20% glycerol, 0.05% bromophenol blue) Environ Sci Pollut Res (2018) 25:8928–8942 8931 at 100 °C for 5 min. SDS-PAGE was performed according to gels. For each set of differentiating spots in replicated gels, Laemmli (1970) using a BioRad MiniProtean System, apply- standard deviation of the fold change was calculated. ing 4% stacking and 10% separating polyacrylamide gels at Three Callitriche extracts obtained upon independent 20-mA per gel. The total of 30 μg protein was loaded per well. physiological experiments were used for proteome mapping. After electrophoresis, the gels were stained with a Coomasie Then, electrophoretic protein profiling of each set of extracts Brillant Blue R250 (Sigma). For calibration of molecular mass (Cr(VI) treatment and control) was done twice, using IPG a BlueEye Prestained Protein Marker (Sigma) set was used. strips of two pH ranges: 3–10 and 5–8. Spot pattern differ- Protein concentration in all extracts and samples was ences were examined for all the replicate gel pairs. In order to determined using a Bradford (1976)methodwithbovinese- properly select only the differentially abundant proteins, we rum albumin (BSA) as a standard. made sure that the candidate spots showed accumulation changes in all of the gel replicates. For subsequent MS/MS analyses, spot excision was made 2DE electrophoresis using the 2DE gels stained without adding glutaraldehyde to the sensitizing solution in order to improve protein identifica- For protein isoelectrofocusing (IEF, the first dimension), the tion. Also, to reduce the number of overlapping proteins, the protein pellet was solubilized in a rehydration buffer (7-M IEF step was performed at pH ranging from 5 to 8. urea, 2-M thiourea, 2% CHAPS, 0.002% bromophenol blue, 20-mM DTT, 1% ampholyte buffer (BioLyte, BioRad)) to a MS/MS protein identification final volume of 150 μl and loaded onto 7-cm IPG strips (BioRad Ready Strip) of the pI range 3–10, and then, in an Protein identification was done at the Institute of independent run, at pI range 5–8. Passive rehydration was Biochemistry and Biophysics of the Polish Academy of carried out for 12 h at 20 °C and the isoelectric focusing was Sciences, Warsaw, Poland. The differentiating spots were ex- performed at 20 °C (Protean IEF Cell, first step 250 V for cised manually and placed in Eppendorf tubes. Then, the gel 20 min, second step 4000 V for 120 min, third step 4000 V, pieces were dried with acetonitrile and subjected to reduction

10,000 V h). Prior to the SDS-PAGE, the IPG strips were with 10-mM DTT in 100-mM NH4HCO3 for 30 min at 57 °C. equilibrated for 10 min in buffer I containing 1% DTT, 6-M Cysteine residues were alkylated with 0.5-M iodoacetamide in urea, 75-mM Tris-HCl, pH 8.8, 30% glycerol, and 2% SDS 100-mM NH4HCO3 (45 min in a darkroom at room tempera- and then, in the second step, for 10 min in buffer II consisting ture) and the proteins were digested overnight with 10-ng/μl of 2.5% iodoacetamide, 6-M urea, 75-mM Tris HCl, pH 8.8, trypsin in 25-mM NH4HCO3 (Promega) at 37 °C. The resul- 30% glycerol, and 2% SDS. The SDS-PAGE (the second di- tant peptide samples were concentrated and desalted on a RP- mension) was performed according to the protocol described C18 precolumn (Waters). Further peptide separation was in the section above using a BioRad Protean II xi Cell 16 × 16- achieved on a nanoultra performance liquid chromatography cm slab unit. In order to achieve maximum reproducibility of (UPLC) RP-C18 column (Waters, BEH130 C18 column, spot patterns for proteome comparison, a pair of IPG strips, 75-μm i.d., 250-mm length) of a nanoACQUITY UPLC sys- obtained upon IEF of either control or Cr-treatment experi- tem, using a 45 min linear acetonitrile gradient. The column ment, were placed side-by-side onto one 16-cm polyacryl- outlet was directly coupled to the electrospray ionization (ESI, amide gel and then overlaid with low melting-point agarose voltage of 1.5 kV) ion source of the Orbitrap Velos type mass (ReadyPrep overlay agarose, BioRad). Protein separation was spectrometer (Thermo), working in the regime of data depen- carried out at 20 mA per gel for about 6 h. After electropho- dent MS to MS/MS switch with HCD type peptide fragmen- resis, the gels were silver-stained as described by Jungblut and tation. A blank run to ensure that there was no cross contam- Seifert (1990), scanned, and digitalized (DNr Bio-Imaging ination from previous samples preceded each analysis. Raw Systems MiniBis Pro, Israel, equipped with a 16-bit, 1.3 data files were preprocessed with Mascot Distiller software Mpix CCD camera). Then, the resultant proteome maps were (version 2.5, MatrixScience). The obtained peptide masses matched to identify differentiating spots. Spot detection and and fragmentation spectra were matched to the National quantification were done automatically employing either the Center Biotechnology Information (NCBI) non-redundant da- Vision Works 2D Lite (UVP) software or Melanie 7.0 tabase no. 20150115 (57,412,064 sequences; 20,591,031,683 (Genebio), followed by manual verification. The amount of residues), with a Viridiplantae filter using the Mascot search protein in each analyzed spot was calculated as a spot pixel engine (Mascot Server v. 2.4.1, MatrixScience) and the density (grayscale) after subtracting gel background value. probability-based algorithm. The following search parameters Then, to compensate for variability of gel staining, protein were applied: enzyme specificity set to trypsin, peptide mass abundance was given as a relative density upon normalization tolerance to ± 30 ppm, and fragment mass tolerance to ± procedure based on the densities of four selected spot marks 0.1 Da. The protein mass was left as unrestricted and mass representing invariant proteins that appeared on all the tested values as monoisotopic with one missed cleavage being 8932 Environ Sci Pollut Res (2018) 25:8928–8942 allowed. Alkylation of cysteine by carbamidomethylation was phosphate buffer, pH 7.0, and 1.8 ml of the buffer. The reac- set as fixed and the oxidation of methionine as a variable tion was initiated with the addition of 200 μl of a sample and modification. Multidimensional protein identification the absorbance decrease was measured at 240 nm after 1 min. technology-type (MudPIT-type) and/or the highest number Superoxide dismutase (SOD) activity was determined accord- of peptide sequences were selected. Mascot peptide ion scores ing to the method of Beauchamp and Fridovich (1971)with served as bases for ranking protein hits. Each ion score was some modifications. The reagent mix was prepared by mixing expressed as – 10 · log(P), where P is the probability that the of 2.18 ml of 100-mM potassium phosphate buffer, pH 7.8, observed match was a random event. Individual ions scores ≥ 0.4 ml of 1.5-mM NBT (nitro blue tetrazolium), 0.2 ml of 55- 43 indicated identity or extensive homology (p <0.05).The mM methionine, 0.2 ml of 0.12-mM riboflavin, and 20 μlofa highest scores in the NCBI database search were matched to sample. The reaction solution was illuminated with a 36-W each analyzed spot. fluorescent lamp for 15 min. A decrease in absorbance was measured spectrophotometrically at the wavelength of Preparation of protein extracts for enzymatic 560 nm. Peroxidases (total activity) were assayed according analyses to Lück (1963). A 2.98-ml volume of 50-mM phosphate buffer, pH 6.2, was mixed with 0.1 ml of 1% p-phenylenediamine

To obtain native protein extracts for zymographic analysis of and 0.1 ml of 0.1% H2O2. The reaction was started with the quinone reductase (QR), 0.8 g of shoots was frozen in liquid addition of 20 μl of the 10-times diluted sample. The absor- nitrogen, ground in a mortar, and suspended in 1.1 ml of bance at 485 nm was measured after 1 and 2 min of incuba- homogenization buffer containing 100-mM Tris-acetate, pH tion. Specific activities of all the above enzymes were 8.0, 100-mM potassium acetate, pH 8.0, 2-mM EDTA, 5-mM expressed as numbers of activity units per mg protein. All DTT, 250-mM sodium ascorbate, and 10% v/v glycerol the assays were done in triplicates. Statistical evaluation was (Laskowski et al. 2002). The samples were centrifuged at performed with the one-way ANOVA variance analysis. 10,000 ×g, 4 °C for 15 min, and the supernatant was collected. Statistical differences between mean values were verified with For activity assays of antioxidant enzymes, 0.8 g of frozen a Tukey post hoc test (α =0.05andn = 3, MATHLAB 2016a shoots was ground in a mortar and extracted with 2 ml of statistical software module). 50-mM Tris-HCl buffer, pH 7.0, containing 1 mM of PMSF. Protein extracts were centrifuged as above. Reagents

Zymographic analysis of quinone reductase (QR) All the chemicals and reagents were of analytical or electrophoretic grades. Nanopure water, IPG strips, agarose, and For zymographic QR assay, a native-PAGE electrophoresis ampholyte solution were from BioRad. Sucrose, potassium was performed at non-denaturing conditions using 10% sepa- acetate, phosphate, sodium ascorbate, sodium chloride, and rating gel and a 4% stacking gel. Samples were mixed with a hydrogen peroxide were obtained from POCh Gliwice, loading buffer containing 0.5-mM Tris-HCl, pH 6.8, 20% Poland. The MS medium was purchased from Duchefa. glycerol, and 0.05% bromophenol blue and then 13 μgof Methylviologen (paraquat) and NBT were from MP protein was loaded per well. Electrophoresis was carried out Biomedicals. All other chemicals and enzymes were pur- at 50 V for 20 min and then at 75 V per gel (Mini Protean chased from Sigma-Aldrich. System, BioRad). The enzymatic staining technique was elaborated based on a QR assay described by Prochaska and Santamaria (1988) and Sharma et al. (1996)withsomemod- Results ifications. The reaction mixture contained 4.5 ml of 0.5-M Tris, 600-μl Tween-20, 60 mg of BSA, 27 mg of MTT In order to study the proteomic response of C. cophocarpa to (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro- Cr(VI), the plants were incubated for 72 h with 1-mM potas- mide), and 90 μl of 50-mM menadione substrate. The reaction sium chromate, which is at conditions evaluated experimen- was launched with 54 μl of 50-mM NADPH. The resultant tally to be sublethal. In independent experiments of this study, gels were digitalized after 60 min of staining. it was established that the treatment caused physiological stress and led to morphological toxicity symptoms covering Assays of enzymatic markers of oxidative stress chlorosis and necrosis of shoots, shortening of apical inter- nodes, and hampered development of apical buds (Fig. 1). Catalase (CAT) activity was measured with a modified spec- Importantly, these effects were reversible since after washing trophotometric method of Aebi (1984) by a decrease of H2O2 out the Cr(VI) solution and further cultivating in the MS me- as observed at 240 nm in a reaction mixture containing 1 ml of dium, full recovery was achieved within several days with no 54-mM solution of hydrogen peroxide in 50-mM potassium manifestations of any disorders. Environ Sci Pollut Res (2018) 25:8928–8942 8933

Fig. 1 Representative photographs of C. cophocarpa apex shoots of control plants and the ones treated for 72 h with 1-mM chromate

Preliminary comparative protein profiling upon chromate treatment was carried out with the SDS-PAGE. The electrophoretic paths show no detectable differences in terms of protein patterns (Fig. 2). Approximately 45 bands can be detected, each revealing similar protein abundances for both control conditions and the Cr(VI)-treated plants. To obtain protein maps with enhanced resolution and protein detectability, two-dimensional Fig. 2 SDS-PAGE protein profiles of shoot extracts of Callitriche cophocarpa treated with 1-mM chromate for 72 h (Cr). C, control electrophoresis was employed with a silver-staining technique. (untreated plant); ST, protein standard markers The resultant protein profiles, as exemplified in Fig. 3aforIPG strips ranging from pH 3 to 10, reveal five repetitive spot differences indicating proteins with altered accumulation. The spots is a flavin mononucleotide-binding, NAD(P)H-dependent have been numbered consecutively and they represent the pro- quinone dehydrogenase that exhibits the in vitro and presum- teins induced de novo (1, 2, marked by green circles) as well as ably also in vivo activity of a quinone reductase (Laskowski the ones down-regulated (3, 4, 5, red circles) upon chromate et al. 2002). Among the other well-scored matches was the treatment. The most pronounced difference can be observed spot no. 2 with MW = 24.2 kDa and pI = 7.3, identified as the for the spot no.1 which was strongly induced only by Cr(VI) light harvesting chlorophyll a/b binding protein (LHCB) from presence and was never detected in the control experiment. That Hedera helix L. This was the second only protein found to be is why we decided to show the surrounding region (marked by a induced upon chromate stress. Spots no. 3 (the observed square in Fig. 3a) in a more detail that is using the IPG strip with values of MW = 58.0 kDa, pI = 6.7) and 4 (MW =44.8kDa, the zoomed pH range of 5–8. The region of interest is presented pI = 7.6) represent down-regulated proteins identified as large in Fig. 3b. The applied narrower pI range allowed much higher RuBisCO subunits from Callitriche hamulata Kütz. and resolution, prevented spot overlapping, and enabled to excise Antirrhinum majus L., respectively. distinct spots, now well separated from the neighboring proteins. Note that for the case of spot no. 4, the initial, randomly made The spots of Fig. 3, qualified as the ones representing differen- peptide scoring yielded unreliable match of a Bhypothetical pro- tiating proteins, were excised from gels and subjected to MS/MS tein PRUPE_ppa008516mg from Prunus persica (L.) Batsch.^ sequencing followed by bioinformatics-based identification. The However, both the protein taxonomic status (Prunus genus dis- results are given in Table 1. tant from Callitriche) and discrepancies between the theoretical

The most important observation is high-ranked identifica- PRUPE molecular mass and pI values (MW =36kDaandpI= tion of the induced protein with a MW = 21.5 kDa and pI = 6.3 9.25) vs. the observed parameters (44 kDa and 6.13, respective- (the observed values, spot no. 1), found to be closely related to ly) gave strong reasons to reject this match. Moreover, the FQR1 dehydrogenase from Vitis vinifera L.. This enzyme PRUPE_ppa008516mg scored very similar to the second-in- 8934 Environ Sci Pollut Res (2018) 25:8928–8942

Fig. 3 2DE proteome mapping of C. cophocarpa shoots after treatment consecutively and marked with circles; spot nos. 1 and 2 represent the with 1-mM potassium chromate for 72 h. a Isoelectric focusing at pI Cr(VI)-induced proteins (green circles), nos. 3–5 indicate the down- range 3–10. b 2DE gel fragment obtained with the pI range of 5–8to regulated proteins (red circles) extend the marked area of the gel (a). Differentiating spots are numbered order protein match of RuBisCO large chain of A. majus and 2). The resultant fragments of protein maps are shown in (Mascot scores of 369 compared to 368, respectively). In addi- Fig. 4c–f. Although they reveal some stress-specific differ- tion, the latter result was characterized by a larger number of ences when compared to the control (Fig. 4a), FQR1 and matching peptides and higher sequence coverage (see Table 1 LHCB were never detected except for the Cr(VI) stress and Supplementary Table 1) and was further supported by sev- (Fig. 4b). This result proves that both proteins of interest were eral other matches with similar Mascot scores indicating induced specifically by the presence of chromate. RuBisCO from other Callitriche species (C. brutia Petagna, In order to check for the QR activity of the chromate- C. cribrosa Schotsman, C. hamulata Kütz., C. platycarpa induced FQR1 protein, extracts from Callitriche shoots prein- Kütz., the results unshown). For the above reasons, the spot cubated with 1 mM Cr(VI) were tested zymographically with no. 4 was finally accepted to represent the RuBisCO large chain the menadione substrate. Figure 5 shows zymograms of protein. For the spot 5, the down-regulated protein identification native-PAGE gels obtained for the control (path C) and as a Bpollen allergen^ of Secale cereale L. is very difficult to Cr(VI)-treated plants (path Cr). There are two major and two interpret and suggests that it is an accidental match caused by a minor bands visible in both paths, and they possibly reflect broad search through the huge database of Viridiplantae. some unspecific menadione-reducing reactions resulting from The chromate treatment proteomic data were compared high content of phenols (see BDiscussion^). However, an ad- with 2D protein profiles of C. cophocarpa shoots subjected ditional zymographic band appeared only within the Cr path to other stressors such as paraquat, hydrogen peroxide, sodi- (indicated with an arrow), thus providing evidence of a newly um chloride, and elevated temperature. At this stage of the produced QR activity occurring in the Cr(VI)-treated plants. study, we focused on the gel region particularly affected by The oxidative stress status of Cr(VI)-treated C. cophocarpa Cr(VI) presence, where the most prominent induction of pro- was evaluated based on activity analyses of selected enzymes teins occurred, that is FQR1 and LHCB (the respective spots 1 known to be responsive to such stress that is peroxidases (Px), Environ Sci Pollut Res (2018) 25:8928–8942 8935 ) d (fold change c Observed b c point (pI) Expression change indicates proteins detected exclusively upon ^ Theoretical c induced B Observed b Theoretical Molecular mass (kDa) Isoelectri deviations. The term Sequence coverage (%) ontrol samples ± standard

Fig. 4 2DE gel fragments revealing C. cophocarpa protein profiles within the area of chromate-induced proteins 1 and 2 (FQR1 and 8 21.2 21.7 21.5 5.8 6.3 Induced Number of matching peptides 5 13.0 50.2 44.8 6.1 7.6 0.5 ± 0.2 25 46.1 50.1 58.0 6.3 6.7 0.8 ± 0.1 3 9.3 20.8 24.2LHCB, 4.8 respectively), 7.3 obtained Induced for different stressors: a control (no stress applied), b Cr(VI) treatment, c paraquat oxidative stress, d hydrogen peroxide oxidative stress, e salt stress, f temperature stress Viridiplantae treated with chromate (1 mM for 72 h) as compared to the control conditions (in vitro incubation). MS/MS protein identification ) 7 30.8 29.8 22.3 6.0 7.7 0.3 ± 0.1 CAT, and SOD. The measurements were carried out at two binding protein b

/ time intervals, which is directly after treatment (3 h) and after a 3-day incubation with a 1-mM chromate solution. The results Secale cereale ) were then compared to the values determined for control ) (untreated) plants. In Fig. 6, it can be clearly seen that ized spot densities as observed in treated and c Callitriche cophocarpa ) Cr(VI) treatment caused over twofold induction of peroxidase(s) activity as measured after 3 h (from 301 ± 38 U/mg protein to 649 ± 45.60 U/mg) and the elevated value was kept after 3-day incubation (572 ± 61.77 U/mg). For CAT, in short- Hedera helix Vitis vinifera Antirrhinum majus Callitriche hamulata ) ( ( ( ( ysis using NCBI databases confined to

Protein name (species) term (3 h) incubation, a strong activity increase was observed

a (0.31 ± 0.06 to 0.81 ± 0.08 U/mg). We note that this effect was

ective sequence record very similar to the one caused by 15-min stressing of Score Callitriche cells directly with hydrogen peroxide (0.79 ± 0.02 U/mg, data not presented). However, the chromate effect on CAT activity was diminished upon long-term incubation and after 3 days, the resultant value was back at the level of Differentiating proteins in proteomes of control (0.35 ± 0.05 U/mg). In the case of SOD, no statistically number significant activity changes were recorded for either 3-h or 3- Calculated from the resp Fold change given as an averaged ratio of normal GivenbyMascot Observed in the SDS-PAGE gel 2 CAA48410 219 Light harvesting chlorophyll Table 1 1 XP_002283286 358 NAD(P)H dehydrogenase (quinone) FQR1 a b c d was followed by bioinformatic anal Cr(VI) treatment 5 CBG76811 276 Pollen allergen Sec c 5 ( 4 Q05554 368 Ribulose bisphosphate carboxylase large chain 3 AAF62288 1951 Ribulose 1,5-bisphosphate carboxylase Spot NCBI accession day treatment with Cr(VI). 8936 Environ Sci Pollut Res (2018) 25:8928–8942

is C. cophocarpa Sendtn. The expected result of this study is to find important protein changes upon chromate treatment, which we believe could explain the unusual capabilities of chromium phytoremediation by C. cophocarpa. Our earlier research work revealed that Cr(VI) presence negatively affected C. cophocarpa shoots leading to several physiological changes such as a decrease of photosynthetic pigment content and a decline of photosynthesis quantum ef- ficiency, distortion of leaf structure, and electrolyte leakage (Augustynowicz et al. 2010, 2013a,b). Moreover, chromate uptake was followed by its rapid reduction (Augustynowicz et al. 2013a, 2016) similarly to the other tested macrophytes Eichhornia crassipes (Mart.) Solms, Salvinia auriculata Aubl., Pistia stratiotes L., and Spirodela polyrhiza (L.) Schleid. (Espinoza-Quiñones et al. 2009; Kaszycki et al. 2005). Unexpectedly, the results of 1D SDS-PAGE protein analyses indicated no visible alterations in the proteome of C. cophocarpa incubated with Cr(VI). Therefore, a more de- Fig. 5 Zymographic analysis of shoot protein extracts of Callitriche tailed study was undertaken employing two-dimensional elec- cophocarpa treated with 1-mM Cr(VI) for 72 h. Enzymatic staining trophoresis, which enabled higher resolution of protein map- with a menadione substrate was done to reveal production of a novel ping as based on pI and MW parameters. 2D electrophoreses quinone reductase activity as indicated by an arrow. C, control yielded significant differences in the resultant protein profiles and showed changes in accumulation of several proteins as Discussion exemplified in Fig. 3. It should be emphasized, however, that many of the protein changes observed for a particular physio- Abiotic stress is known to alter proteomic patterns in many logical experiment might not be linked directly to the chro- plants (Jorrín-Novo et al. 2009). Comparative proteomics mate stress but rather to indigenous proteome pattern variabil- analyses can be a source of important novel information on ity, which is typical of 2DE-based profiling. This variability the nature of stress tolerance and adaptation mechanisms and may result from the noisiness of gene expression (Chalancon may suggest occurrence of newly induced biochemical path- et al. 2012), intrinsic physicochemical diversity of proteins, ways (Ahsan et al. 2009;Kosováetal.2011; Timperio et al. and protein abundance differences between individual plant 2008). For studies of heavy metal interactions with assimila- objects (Chandramouli and Qian 2009; Jorrín-Novo et al. tive plant organs, aquatic plants are regarded as favorable 2009;Lopez2007). The above reasons made us pick out only models. Macrophytes are also preferable candidates for effi- these spots whose densities changed in all of the gel pair cient phytoremediation in aqueous environments (Parvaiz replicates. Such an approach involving stringent conditions 2016). for selecting differentially abundant proteins enabled us to So far, many scientific contributions have been published, detect five protein alterations qualified as chromate stress spe- in which heavy metals were shown to affect plant proteomes cific for C. cophocarpa treated with 1-mM Cr(VI) for 72 h (for reviews see DalCorso et al. 2013;Kosováetal.2011; (Table 1). Ovečka and Takáč 2014; Visioli and Marmiroli 2013). In proteomic studies, the number of differentiating proteins However, the available proteomic reference data regarding in plants exposed to environmental stressors may vary in a chromium effect on plants is relatively poor and deal predom- broad range of several to several hundred detected spots inately with terrestrial plant models (Cvjetko et al. 2014; (Kosová et al. 2011). In our recently launched experiments Hossain and Komatsu 2013; Vannini et al. 2011; Yildiz and on 72-h chromate treatment of three other tested submerged Terzi 2016; Zemleduch-Barylska and Lorenc-Plucińska macrophytes (S. polyrhiza, Elodea canadensis Michx., and 2015). To our knowledge, there are no proteomic reports on Lemna trisulca L.), we revealed the total of 13, 12, and 21 aquatic plant-chromium interactions except that of Bah et al. differentially abundant proteins, respectively (Kaszycki et al., (2010) which brings comparative data on Cr, Cd, and Pb in- manuscript in preparation). It should be pointed out here that, fluence on Typha angustifolia L. protein profiles. For the besides methodological constraints of 2DE (DalCorso et al. above reasons, the presented work is the first one to provide 2013; Jorrín-Novo et al. 2009; Lopez 2007; Visioli and evidence on chromate stress-induced proteome alterations in a Marmiroli 2013), the resultant protein profile of abiotically macrophyte with entirely submerged assimilative organs that stressed plant strongly depends on the time scale of Environ Sci Pollut Res (2018) 25:8928–8942 8937

Fig. 6 Specific activities of oxidative stress enzyme markers: Px, peroxidase; CAT, catalase; SOD, superoxide dismutase, obtained for shoot extracts of Callitriche cophocarpa subjected to chromate treatment for 3 (light- gray bars) and 72 h (3 days, dark- gray bars). White bars: control experiment (untreated shoots). The mean values of particular enzyme activities (n =3±SD) marked with different letters are significantly different for p ˂ 0.05 8938 Environ Sci Pollut Res (2018) 25:8928–8942 observation. Some of the early-response changes including three selected antioxidant enzymes were assayed, i.e., SOD oxidative stress reactions can be observed in a matter of hours (EC 1.15.1.1), CAT (EC 1.11.1.6), and (a sum of) peroxidases (Kosová et al. 2011; Singh et al. 2013), whereas enhanced (Px). These enzymes are known as oxidative stress markers in proteomic rearrangements are typically best pronounced with- plants (Geebelen et al. 2002) and were reported earlier to be in 3–5 days, which is the time required for a plant to evolve an responsive to chromate (Shanker et al. 2005).SODactsasa altered phenotype due to gene induction and protein biosyn- scavenger of the superoxide anion, whereas CAT and Pxs cat- thesis (Forni et al. 2012;Kosováetal.2011;Zhaoetal.2005). alytically eliminate hydrogen peroxide. At first, a 3-h treatment In turn, in longer-term experiments, mechanisms of plant ac- was examined, which revealed direct reaction to Cr(VI) by climation and adaptation to harsh conditions (see Bah et al. strongly increasing CAT and peroxidase activities. This early- 2010 for an example) may lead to further differences in pro- response stage (an alarm phase of plant response as indicated teomic profiles. by Kosová et al. 2011) is consistent with our previous obser- The proteomic changes observed in this study (see Table 1) vations which showed immediate reaction of C. cophocarpa can account for several important aspects of physiologically shoots treated with chromate and manifested by a rapid (in a altered phenotype of C. cophocarpa under chromate stress. matter of hours) reduction of Cr(VI) to the Cr(V) intermediate The case of protein spot 1 (a putative FQR1 enzyme) is which is generated upon Cr(VI) → Cr(III) bioconversion discussed below in a more detail since it has possible direct (Augustynowicz et al. 2013a, 2016). During prolonged (3- consequences in terms of chromate resistance and bioremedia- day) incubation, only Px activities were kept elevated while tion. The light harvesting chlorophyll a/b binding protein CAT was back at the level determined for control plants. (LHCB), identified in spot 2, was strongly induced by SOD, CAT, and peroxidase induction and/or activation are Cr(VI). This is an abundant membrane apoprotein of photosys- known to play a major role in heavy metal detoxication in tem II (PSII) and is normally complexed with chlorophyll and plants (Ahsan et al. 2009). However, in plants treated with xanthophylls to serve as the antenna complex (Jansson 1999). chromate, as reviewed by Shanker et al. (2005) and Singh Importantly, the expression of the LHCB gene was found to et al. (2013), the activity of these enzymes increased only at depend upon environmental conditions including, among relatively low chromate concentrations (usually of the order of others, oxidative stress (Xu et al. 2012). Therefore, the induc- micromoles per liter) whereas the excess of Cr(VI) (most typ- tion of this protein suggests its role in Cr bioremediation; how- ically above 100 μM) led to significant activity inhibition. The ever, at this stage, it is difficult to propose a particular mecha- fact that in C. cophocarpa theactivityofbothSODandCAT nism. So far, LHCB proteins were found to be either up- remained at control levels under harsh conditions of 3-day 1- regulated by chromate in a Cr(VI)-tolerant canola (Brassica mM Cr(VI) presence, it brings additional evidence that the napus L.) cultivar (Yildiz and Terzi 2016) or inhibited in a studied macrophyte could induce enhanced resistance to oxi- Cr(VI)-hypersensitive microphyte Pseudokirchneriella dative stress. In the context of the above, the mechanism of subcapitata (Korshikov) F. Hindák (Vannini et al. 2009). long-term activation of peroxidases under chromate treatment RuBisCO (ribulose bisphosphate carboxylase), the enzyme needs further research. These enzymes were previously shown catalyzing a key reaction of carbon dioxide fixation in photo- to be efficient scavengers of heavy metal borne H2O2 (Ahsan synthesis, was down-regulated (spots 3 and 4). This was an et al. 2009). However, the increase of Px activities as observed expected result since chromate at 1-mM concentration was in enzymatic assays was not reflected in 2DE analyses by shown to inhibit photosynthesis of C. cophocarpa elevated abundance of any known peroxidase. This result (Augustynowicz et al. 2010, 2013b). RuBisCO down- can be explained by the stimulation of enzymatic activity with regulation was shown in many plant models and can be biochemical regulatory mechanisms rather than by induced regarded as a typical response to abiotic stress including heavy expression. Similar discrepancies were reported by other au- metal treatments (Ahsan et al. 2009; Kosová et al. 2011). thors who detected peroxidase activity stimulation by Cr Accordingly and similarly to our observations, its accumula- (Zemleduch-Barylska and Lorenc-Plucińska 2015)orCd tion decreased upon 3-day chromate incubation of both canola (e.g., Yang et al. 2015; Kieffer et al. 2009) but observed no (Yildiz and Terzi 2016) and P. subcapitata (Vannini et al. accumulation changes in 2DE profiling. 2009); however, under conditions of long-term (30-day) ad- Taken the above findings together, it appears that aptation to Cr(VI), the enzyme was significantly up-regulated C. cophocarpa shoots can adapt to 1-mM Cr(VI) within 3 days in Typha angustifolia (Bah et al. 2010). by increasing activity of peroxidases while maintaining the Since Cr(VI) treatment is typically associated with the oxi- levels of the other antioxidant enzymes: SOD and CAT. dative stress caused by elevated levels of ROS (see This, in turn, indicates the tendency to suppress oxidative BIntroduction^), it was of interest to verify whether stress and suggests that chromate presence can launch protec- C. cophocarpa triggered any relevant enzymatic radical- tive mechanisms resulting in lowered pool of toxic ROS. scavenging mechanisms and whether it could adapt during a The FQR1 protein as identified in spot 1 of the electropho- prolonged (3-day) incubation with Cr(VI). The activities of retic gel (the highest score of V. vinifera NAD(P)H quinone Environ Sci Pollut Res (2018) 25:8928–8942 8939 dehydrogenase, see Fig. 3 and Table 1) was shown to be very observed in Callitriche. Cr(VI) biological reduction process strongly and specifically induced by chromate. Its presence has been documented for plants as well as for all other organ- was always well pronounced only in Cr-treated plants whereas isms: pro- and eukaryotic microbes, fungi, algae, and animals both under control conditions and in all cases of other stressors (Joutey et al. 2015; Singh et al. 2013;Zhitkovich2011). In (Fig. 4a, c–f), the electrophoretic gels lacked accumulation of general, hexavalent chromate reduction may involve both any protein related to FQR1. A closer look at this enzyme extra- and intracellular processes and both enzymatic and enables us to suggest its direct involvement in chromate bio- non-enzymatic mechanisms. For Cr(VI) bioconversion inside remediation as a putative quinone and/or chromate reductase. cell, the reduction process may have two contradictory as- Extensive database search (www.ebi.ac.uk/interpro/protein/ pects: first, it can result in cellular toxicity due to oxidative Q9LSQ5, accession date: July 2017) following the NCBI stress, and second, it can serve as a bioremediation (resistance) protein identification accessions gave more information on mechanism. In the first case, single-electron reactions lead to the enzyme identity. The FQR1 domain is not unique and generation of detrimental ROS (Cheung and Gu, 2007; can be found in several flavoproteins. It belongs to a big Zhitkovich 2011; Thatoi et al. 2014) which in turn induce family of highly conserved flavin-binding reductases occur- antioxidative metabolism producing cellular antioxidants ring in plants, fungi, archaea, and eubacteria and shares some (Panda and Choudhury 2005; Singh et al. 2013). In the second homologies with mammalian quinone reductases (Laskowski case, the cellular Cr(VI) → Cr(III) conversion is achieved et al. 2002). In particular, a NAD(P)H:quinone oxidoreductase preferably via a two-electron transfer systems which enable can reduce quinones to the hydroquinone state in a two- an organism to cope with the chromate stress by safely (that is electron reaction, which prevents the interaction of the minimizing ROS production) reducing the original hexavalent semiquinone with O2 and thus disables production of toxic form to the less mobile and thus less reactive Cr(III) (Ramírez- superoxide. So, the enzyme has been postulated to function Díaz et al. 2008;Jouteyetal.2015). as a protective agent in oxidative stress response and to par- Several enzymes capable of Cr(VI) reduction have been ticipate in detoxifying reactions (Berczi and Moller 2000; described, first identified in bacteria where various chromate Laskowski et al. 2002). reductases were shown to influence microbial resistance to Zymographic analyses (see the enzymatic band indicated Cr(VI) (Ramírez-Díaz et al. 2008; Thatoi et al. 2014 and the by an arrow in Fig. 5) made it possible to prove that the QR references therein). Note that these reported chromate- activity was indeed induced in Callitriche treated with chro- reducing activities relied upon either one- or two-electron mate. To detect an in vitro QR activity in Cr(VI)-stressed mechanisms, which resulted in different strain sensitivities to plants, we used a modified assay enabling to visualize the chromate as discussed above. Importantly, the two-electron enzyme in native-PAGE gels. Such an approach was neces- Cr(VI) reduction systems that enable safer Cr(VI) detoxica- sary since the standard laboratory assays (data unshown) tion were typically linked to soluble flavin mononucleotide- failed to bring conclusive results, yielding high apparent binding oxidoreductases producing QR activities. These en- (background) activities, possibly due to high abundance of zymes were induced by chromate and identified as bacterial phenolics in C. cophocarpa (Augustynowicz et al. 2014a). chromate reductases because they could use Cr(VI) as a pos- It should be emphasized here that QR activities in Cr(VI)- sible terminal electron acceptor (Eswaramoorthy et al. 2012; stressed plants have not been reported to date. Although qui- Thatoi et al. 2014). none oxidoreductases are ubiquitous among plant species, there Besides bacteria, Cr(VI)-reducing activities were also ob- are only a few proteomic studies providing evidence of QRs served in yeast, microalgae, fungi (Joutey et al. 2015; Viti being responsive to other heavy metals or metalloids. First, et al. 2014), as well as plants (Prado et al. 2016; Shanker Requejo and Tena (2005) showed a p-benzoquinone reductase et al. 2009) including macrophytes (Espinoza-Quiñones (bQR) induction in a maize (Zea mays L.) root proteome treated et al. 2009;Kaszyckietal.2005)andC. cophocarpa in par- with arsenic and proposed a protective role of the enzyme ticular (Augustynowicz et al. 2013a, 2016). Importantly, for against oxidative stress. For cadmium-stressed plants, Kieffer the latter case, chromate bioreduction occurred exclusively et al. (2009) reported strong up-regulation of FQR1 as well as within the plant shoots and was not observed outside the mac- several other bQR-like and putative QR proteins in poplar rophyte tissue (that is in a surrounding growth medium). This leaves. Zhao et al. (2011), in turn, revealed enhanced accumu- result was based on a low-frequency in vivo electron paramag- lation of bQR in a leaf proteome of a Cd-hyperaccumulator netic resonance study (Augustynowicz et al. 2013a) which Phytolacca americana L. Recently, Chen et al. (2015) detected revealed that C. cophocarpa chromate phytoremediation in- a protein described as a Bputative quinone reductase 2^ in the volved Cr(VI) intratissular reductive conversion generating a proteome of Cu-stressed rice (Oryza sativa L.). Cr(V) intermediate. There is scientific evidence enabling us to hypothesize that As regards plants, chromate reductases have not been iden- the induction of the newly identified FQR1 enzyme plays an tified in vivo, yet. However, in a study on Arabidopsis important role in the mechanism of chromate bioreduction as thaliana (L.) Heynh., Sparla et al. (2003)documentedthata 8940 Environ Sci Pollut Res (2018) 25:8928–8942 flavoenyzme NAD(P)H:quinone oxidoreductase (NQR) Acknowledgements We greatly appreciate helpful discussions with Prof. B ^ shared sequence homologies with some bacterial chromate Cinzia Forni (Università di Roma Tor Vergata , Italy) regarding stress reactions of aquatic plants and with Agata Malinowska, M.Sc. reductases. This led the authors to the idea that the enzyme (Laboratory of Mass Spectrometry, Institute of Biochemistry and could function as a chromate reductase and thus promote Cr Biophysics, Polish Academy of Sciences), regarding Mascot data inter- phytoremediation. Using A. thaliana homogenates, they de- pretation. We are also indebted to Dr. Magdalena Kwolek-Mirek ł tected a NQR-based NADPH-chromate reductase activity in (University of Rzeszów, Poland) and Dr. Anna Ko ton (University of Agriculture, Kraków, Poland) for their advice on oxidative stress induc- in vitro tests. Note however, that the Arabidopsis NQR pro- tion and monitoring in C. cophocarpa. tein, unlike the enzyme identified in this study, was not induc- ible and it belonged to a different family of quinone reduc- Funding information The work was financially supported by the research tases, poorly related to the FQR domains (Heyno et al. 2013). grant of the Polish National Science Centre No. DEC-2011/03/B/NZ9/ 00952. The equipment used for MS-based protein identification was in Later, Shanker et al. (2009), based on extensive bioinformatic part sponsored by the Centre for Preclinical Research and Technology database search, speculated that putative plant chromate re- (CePT), a project supported by the European Regional Development ductases homologous to the microbial ones might take part Fund and Innovative Economy, the National Cohesion Strategy of in Cr(VI) detoxification. Poland (http://www.ibb.waw.pl/en/services/mass-spectrometry-lab). Taking all the above facts into consideration it is tempting Open Access This article is distributed under the terms of the Creative to suggest that C. cophocarpa FQR1 quinone reductase shows Commons Attribution 4.0 International License (http:// chromate-reducing activity and is an important factor involved creativecommons.org/licenses/by/4.0/), which permits unrestricted use, in Cr(VI) bioremediation. However, the postulated role of the distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link enzyme still awaits direct experimental evidence. If the novel to the Creative Commons license, and indicate if changes were made. enzyme proved to reduce Cr(VI) substrate both in vitro and in vivo, its induction with chromate would indicate that C. cophocarpa is the first known aquatic plant to have devel- References oped some specific enzymatic detoxifying mechanism against Cr(VI) action. This, in turn, would imply that our plant model Aebi H (1984) Catalase in vitro. 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